Targeting Notch signaling with a Notch2/Notch3 antagonist (tarextumab) inhibits tumor growth and decreases tumor-initiating cell frequency.

PURPOSE: The Notch pathway plays an important role in both stem cell biology and cancer. Dysregulation of Notch signaling has been reported in several human tumor types. In this report, we describe the development of an antibody, OMP-59R5 (tarextumab), which blocks both Notch2 and Notch3 signaling. EXPERIMENTAL DESIGN: We utilized patient-derived xenograft tumors to evaluate antitumor effect of OMP-59R5. Immunohistochemistry, RNA microarray, real-time PCR, and in vivo serial transplantation assays were employed to investigate the mechanisms of action and pharmacodynamic readouts. RESULTS: We found that anti-Notch2/3, either as a single agent or in combination with chemotherapeutic agents was efficacious in a broad spectrum of epithelial tumors, including breast, lung, ovarian, and pancreatic cancers. Notably, the sensitivity of anti-Notch2/3 in combination with gemcitabine in pancreatic tumors was associated with higher levels of Notch3 gene expression. The antitumor effect of anti-Notch2/3 in combination with gemcitabine plus nab-paclitaxel was greater than the combination effect with gemcitabine alone. OMP-59R5 inhibits both human and mouse Notch2 and Notch3 function and its antitumor activity was characterized by a dual mechanism of action in both tumor and stromal/vascular cells in xenograft experiments. In tumor cells, anti-Notch2/3 inhibited expression of Notch target genes and reduced tumor-initiating cell frequency. In the tumor stroma, OMP-59R5 consistently inhibited the expression of Notch3, HeyL, and Rgs5, characteristic of affecting pericyte function in tumor vasculature. CONCLUSIONS: These findings indicate that blockade of Notch2/3 signaling with this cross-reactive antagonist antibody may be an effective strategy for treatment of a variety of tumor types.

Blood-brain barrier KCa3.1 channels: evidence for a role in brain Na uptake and edema in ischemic stroke.

BACKGROUND AND PURPOSE: KCa3.1, a calcium-activated potassium channel, regulates ion and fluid secretion in the lung and gastrointestinal tract. It is also expressed on vascular endothelium where it participates in blood pressure regulation. However, the expression and physiological role of KCa3.1 in blood-brain barrier (BBB) endothelium has not been investigated. BBB endothelial cells transport Na(+) and Cl(-) from the blood into the brain transcellularly through the co-operation of multiple cotransporters, exchangers, pumps, and channels. In the early stages of cerebral ischemia, when the BBB is intact, edema formation occurs by processes involving increased BBB transcellular Na(+) transport. This study evaluated whether KCa3.1 is expressed on and participates in BBB ion transport. METHODS: The expression of KCa3.1 on cultured cerebral microvascular endothelial cells, isolated microvessels, and brain sections was evaluated by Western blot and immunohistochemistry. Activity of KCa3.1 on cerebral microvascular endothelial cells was examined by K(+) flux assays and patch-clamp. Magnetic resonance spectroscopy and MRI were used to measure brain Na(+) uptake and edema formation in rats with focal ischemic stroke after TRAM-34 treatment. RESULTS: KCa3.1 current and channel protein were identified on bovine cerebral microvascular endothelial cells and freshly isolated rat microvessels. In situ KCa3.1 expression on BBB endothelium was confirmed in rat and human brain sections. TRAM-34 treatment significantly reduced Na(+) uptake, and cytotoxic edema in the ischemic brain. CONCLUSIONS: BBB endothelial cells exhibit KCa3.1 protein and activity and pharmacological blockade of KCa3.1 seems to provide an effective therapeutic approach for reducing cerebral edema formation in the first 3 hours of ischemic stroke.

Ischemic factor-induced increases in cerebral microvascular endothelial cell Na/H exchange activity and abundance: evidence for involvement of ERK1/2 MAP kinase.

Brain edema forms rapidly in the early hours of ischemic stroke by increased secretion of Na, Cl, and water into the brain across an intact blood-brain barrier (BBB), together with swelling of astrocytes as they take up the ions and water crossing the BBB. Our previous studies provide evidence that luminal BBB Na-K-Cl cotransport (NKCC) and Na/H exchange (NHE) participate in ischemia-induced edema formation. NKCC1 and two NHE isoforms, NHE1 and NHE2, reside predominantly at the luminal BBB membrane. NKCC and NHE activities of cerebral microvascular endothelial cells (CMEC) are rapidly stimulated by the ischemic factors hypoxia, aglycemia, and AVP, and inhibition of NKCC and NHE activities by bumetanide and HOE642, respectively, reduces brain Na uptake and edema in the rat middle cerebral artery occlusion model of stroke. The present study was conducted to further explore BBB NHE responses to ischemia. We examined whether ischemic factor-stimulated NHE activity is sustained over several hours, when the majority of edema forms during stroke. We also examined whether ischemic factors alter NHE1 and/or NHE2 protein abundance. Finally, we conducted initial studies of ERK1/2 MAP kinase involvement in BBB NHE and NKCC responses to ischemic factors. We found that hypoxia, aglycemia, and AVP increase CMEC NHE activity through 5 h and that NHE1, but not NHE2, abundance is increased by 1- to 5-h exposures to these factors. Furthermore, we found that these factors rapidly increase BBB ERK1/2 activity and that ERK1/2 inhibition reduces or abolishes ischemic factor stimulation of NKCC and NHE activities.

Ischemia-induced stimulation of cerebral microvascular endothelial cell Na-K-Cl cotransport involves p38 and JNK MAP kinases.

Previous studies have provided evidence that, in the early hours of ischemic stroke, a luminal membrane blood-brain barrier (BBB) Na-K-Cl cotransporter (NKCC) participates in ischemia-induced cerebral edema formation. Inhibition of BBB NKCC activity by intravenous bumetanide significantly reduces edema and infarct in the rat permanent middle cerebral artery occlusion model of ischemic stroke. We demonstrated previously that the BBB cotransporter is stimulated by hypoxia, aglycemia, and AVP, factors present during cerebral ischemia. However, the underlying mechanisms have not been known. Ischemic conditions have been shown to activate p38 and JNK MAP kinases (MAPKs) in brain, and the p38 and JNK inhibitors SB-239063 and SP-600125, respectively, have been found to reduce brain damage following middle cerebral artery occlusion and subarachnoid hemorrhage, respectively. The present study was conducted to determine whether one or both of these MAPKs participates in ischemic factor stimulation of BBB NKCC activity. Cultured cerebral microvascular endothelial cell NKCC activity was evaluated as bumetanide-sensitive (86)Rb influx. Activities of p38 and JNK were assessed by Western blot and immunofluorescence methods using antibodies that detect total vs. phosphorylated (activated) p38 or JNK. We report that p38 and JNK are present in cultured cerebral microvascular endothelial cells and in BBB endothelial cells in situ and that hypoxia (7% O(2) and 2% O(2)), aglycemia, AVP, and O(2)-glucose deprivation (5- to 120-min exposures) all rapidly activate p38 and JNK in the cells. We also provide evidence that SB-239063 and SP-600125 reduce or abolish ischemic factor stimulation of BBB NKCC activity. These findings support the hypothesis that ischemic factor stimulation of the BBB NKCC involves activation of p38 and JNK MAPKs.

Ischemia-induced stimulation of Na-K-Cl cotransport in cerebral microvascular endothelial cells involves AMP kinase.

Increased blood-brain barrier (BBB) Na-K-Cl cotransporter activity appears to contribute to cerebral edema formation during ischemic stroke. We have shown previously that inhibition of BBB Na-K-Cl cotransporter activity reduces edema and infarct in the rat middle cerebral artery occlusion (MCAO) model of ischemic stroke. We have also shown that the BBB cotransporter is stimulated by the ischemic factors hypoxia, aglycemia, and arginine vasopressin (AVP), although the mechanisms responsible are not well understood. AMP-activated protein kinase (AMPK), a key mediator of cell responses to stress, can be activated by a variety of stresses, including ischemia, hypoxia, and aglycemia. Previous studies have shown that the AMPK inhibitor Compound C significantly reduces infarct in mouse MCAO. The present study was conducted to evaluate the possibility that AMPK participates in ischemic factor-induced stimulation of the BBB Na-K-Cl cotransporter. Cerebral microvascular endothelial cells (CMEC) were assessed for Na-K-Cl cotransporter activity as bumetanide-sensitive (86)Rb influx. AMPK activity was assessed by Western blot analysis and immunofluorescence methods using antibodies that detect total versus phosphorylated (activated) AMPK. We found that hypoxia (7% and 2% O(2)), aglycemia, AVP, and oxygen-glucose deprivation (5- to 120-min exposures) increase activation of AMPK. We also found that Compound C inhibition of AMPK reduces hypoxia-, aglycemia-, and AVP-induced stimulation of CMEC Na-K-Cl cotransporter activity. Confocal immunofluorescence of perfusion-fixed rat brain slices revealed the presence of AMPK, both total and phosphorylated kinase, in BBB in situ of both control and ischemic brain. These findings suggest that ischemic factor stimulation of the BBB Na-K-Cl cotransporter involves activation of AMPK.

Effects of estradiol on ischemic factor-induced astrocyte swelling and AQP4 protein abundance.

In the early hours of ischemic stroke, cerebral edema forms as Na, Cl, and water are secreted across the blood-brain barrier (BBB) and astrocytes swell. We have shown previously that ischemic factors, including hypoxia, aglycemia, and arginine vasopressin (AVP), stimulate BBB Na-K-Cl cotransporter (NKCC) and Na/H exchanger (NHE) activities and that inhibiting NKCC and/or NHE by intravenous bumetanide and/or HOE-642 reduces edema and infarct in a rat model of ischemic stroke. Estradiol also reduces edema and infarct in this model and abolishes ischemic factor stimulation of BBB NKCC and NHE. There is evidence that NKCC and NHE also participate in ischemia-induced swelling of astrocytes. However, little is known about estradiol effects on astrocyte cell volume. In this study, we evaluated the effects of AVP (100 nM), hypoxia (7.5% O(2)), aglycemia, hypoxia (2%)/aglycemia [oxygen glucose deprivation (OGD)], and estradiol (1-100 nM) on astrocyte cell volume using 3-O-methyl-d-[(3)H]glucose equilibration methods. We found that AVP, hypoxia, aglycemia, and OGD (30 min to 5 h) each significantly increased astrocyte cell volume, and that estradiol (30-180 min) abolished swelling induced by AVP or hypoxia, but not by aglycemia or OGD. Bumetanide and/or HOE-642 also abolished swelling induced by AVP but not aglycemia. Abundance of aquaporin-4, known to participate in ischemia-induced astrocyte swelling, was significantly reduced following 7-day but not 2- or 3-h estradiol exposures. Our findings suggest that hypoxia, aglycemia, and AVP each contribute to ischemia-induced astrocyte swelling, and that the edema-attenuating effects of estradiol include reduction of hypoxia- and AVP-induced astrocyte swelling and also reduction of aquaporin-4 abundance.